This invention relates to a novel heteromultimer and its use in screening pharmaceutically active compounds for modulators of maxi-K channel activity. Such modulators are useful in treating asthma, pregnant human myometrium, cerebral ischemia and in conditions where stimulation of neurotransmitter release is desired such as Alzheimer's disease and stimulation of damaged nerves.
The present invention relates to the combined use of both the .alpha. and .beta. subunit of a mammalian calcium-activated potassium channel originally identified and separated from bovine tracheal smooth muscle, which confers pharmacological properties to the .alpha. and .beta. subunit complex similar to that found with the native channel. It further relates to the use of the .alpha.-.beta. heteromultimer in expression systems as assays for agonists or antagonists of calcium-activated potassium channels.
Potassium channel antagonists are useful for a number of physiological disorders in mammals, including humans. Ion channels, including potassium channels, are found in all mammalian cells and are involved in the modulation of various physiological processes and normal cellular homeostasis. Potassium channels generally control the resting membrane potential, and the efflux of potassium ions causes repolarization of the plasma membrane after cell depolarization. Potassium channel antagonists prevent repolarization and cause the cell to stay in the depolarized, excited state.
There are a number of potassium channel subtypes. Physiologically, one important subtype is the maxi-K channel, defined as high -conductance calcium-activated potassium channel, which is present in neuronal tissue and smooth muscle. Intracellular calcium concentration (Ca.sup.2+.sub.i) and membrane potential gate these channels. For example, maxi-K channels are opened to enable efflux of potassium ions by an increase in the intracellular Ca.sub.2+ concentration or by membrane depolarization (change in potential). Elevation of intracellular calcium concentration is required for neurotransmitter release, smooth muscle contraction, proliferation of some cell types and other processes. Modulation of maxi-K channel activity therefore affects cellular processes that depend on influx of calcium through voltage-dependent pathways, such as transmitter release from the nerve terminals and smooth muscle contraction. The screening procedures revealed by the present invention are therefore useful for detecting compounds with utility in the treatment of neurological disorders in which neurotransmitter release is impaired.
A number of marketed drugs function as potassium channel antagonists. The most important of these include the compounds Glyburide, Glipizide and Tolbutamide. These potassium channel antagonists are useful as antidiabetic agents. Potassium channel antagonists are also utilized as Class III antiarrhythmic agents and to treat acute infractions in humans. A number of naturally occurring toxins are known to block potassium channels including apamin, iberiotoxin, charybdotoxin, margatoxin, noxiustoxin, kaliotoxin, dendrotoxin(s), mast cell degranuating (MCD) peptide, and .beta.-bungarotoxin (.beta.-BTX).
Depression is related to a decrease in neurotransmitter release. Current treatments of depression include blockers of neurotransmitter uptake, and inhibitors of enzymes involved in neurotransmitter degradation which act to prolong the lifetime of neurotransmitters.
It is believed that certain diseases such as depression, memory disorders and Alzheimer's disease are the result of an impairment in neurotransmitter release. Potassium channel antagonists may therefore be utilized as cell excitants which should stimulate release of neurotransmitters such as acetylcholine, serotonin and dopamine. Enhanced neurotransmitter release should reverse the symptoms associated with depression and Alzheimer's disease.
The present invention relates to the use of the calcium activated potassium channel .alpha. and .beta. subunits in transient or stable coexpression systems as assays for antagonists of maxi-K channels. Such blockers are useful in diseases where neurotransmission is deficient, such as Alzheimer's or depression.
The present invention also relates to the use of the .alpha. and .beta. subunits in transient or stable coexpression systems as assays to screen for agonists of maxi-K channels. Such agonists are useful in diseases involving excessive smooth muscle tone or excitability such as asthma, angina, hypertension, incontinence, pre-term labor, migraine, cerebral ischemia and irritable bowl syndrome. Such agonists also act to decrease neurotransmitter or hormone release, and thus are of use in treating diseases such as asthma, cerebral ischemia and pain modulation. Specifically such agents can act to decrease release of tachykinins, such as substance P and neurokinin A, among others, that are involved in the neurogenic intimation that occurs in asthma. Thus, agonists of maxi-K channels would be expected to decrease neurogenic inflamation and be useful in the treatment of asthma.
Agonists of maxi-K channels hyperpolarize neurons and thereby, decrease calcium entry through both voltage-dependent calcium channels and through excitatory neurotransmitter activated channels. Since elevation of intracellular calcium is part of the process which leads to cell damage, reduction in calcium entry would decrease neural damage in cerebral ischemia.